The third cytoplasmic loop of the angiotensin II type 1 receptor exerts differential effects on extracellular signal-regulated kinase (ERK1/ERK2) and apoptosis via Ras- and Rap1-dependent pathways

Judith Haendeler, Mari Ishida, L. Hunyady, Bradford C. Berk

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Abstract

The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT1) is important for receptor coupling to G proteins and activation of downstream events. Therefore, we determined whether specific AT1 sequences were required for kinase activation and inhibition of apoptosis by transfecting wild-type (AT1Rwt) and mutated AT1 into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal-regulated kinase (ERK1/ERK2) activity in 293 cells transfected with AT1Rwt. However, in 293 cells that expressed a receptor in which amino acids 221 and 222 were deleted (AT1R[Del221/222]), Ang II-mediated ERK1/ERK2 activation was inhibited by >85%. In contrast, c-Jun NH2-terminal protein kinase (JNK) activation was similar in AT1Rwt- and AT1R(Del221/222)-transfected cells. Activation of ERK1/ERK2 by AT1Rwt was independent of Ca2+, whereas the low level of ERK1/ERK2 activation by AT1R(Del221/222) was completely Ca2+ dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(Del221/222) required Rap1. These results demonstrate the presence of 2 different pathways for ERK1/ERK2 activation by Ang II, which differ in their requirements for Ca2+ and small G proteins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation-induced apoptosis in cells transfected with AT1Rwt but not AT1R(Del221/222). AKT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpression of constitutively active AKT significantly reduced serum deprivation-induced apoptosis in cells transfected with AT1R(Del221/222). This study shows for the first time a direct link between kinase activation and inhibition of apoptosis dependent on amino acids 221 and 222 in the third cytoplasmic loop of the AT1.

Original languageEnglish
Pages (from-to)729-736
Number of pages8
JournalCirculation Research
Volume86
Issue number7
Publication statusPublished - Apr 14 2000

Fingerprint

Angiotensin Type 1 Receptor
Extracellular Signal-Regulated MAP Kinases
Apoptosis
Angiotensin II
MAP Kinase Signaling System
Phosphotransferases
Amino Acids
Monomeric GTP-Binding Proteins
JNK Mitogen-Activated Protein Kinases
Serum
GTP-Binding Proteins
Protein Kinases

Keywords

  • Angiotensin II
  • Apoptosis
  • AT
  • Kinases

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

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title = "The third cytoplasmic loop of the angiotensin II type 1 receptor exerts differential effects on extracellular signal-regulated kinase (ERK1/ERK2) and apoptosis via Ras- and Rap1-dependent pathways",
abstract = "The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT1) is important for receptor coupling to G proteins and activation of downstream events. Therefore, we determined whether specific AT1 sequences were required for kinase activation and inhibition of apoptosis by transfecting wild-type (AT1Rwt) and mutated AT1 into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal-regulated kinase (ERK1/ERK2) activity in 293 cells transfected with AT1Rwt. However, in 293 cells that expressed a receptor in which amino acids 221 and 222 were deleted (AT1R[Del221/222]), Ang II-mediated ERK1/ERK2 activation was inhibited by >85{\%}. In contrast, c-Jun NH2-terminal protein kinase (JNK) activation was similar in AT1Rwt- and AT1R(Del221/222)-transfected cells. Activation of ERK1/ERK2 by AT1Rwt was independent of Ca2+, whereas the low level of ERK1/ERK2 activation by AT1R(Del221/222) was completely Ca2+ dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(Del221/222) required Rap1. These results demonstrate the presence of 2 different pathways for ERK1/ERK2 activation by Ang II, which differ in their requirements for Ca2+ and small G proteins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation-induced apoptosis in cells transfected with AT1Rwt but not AT1R(Del221/222). AKT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpression of constitutively active AKT significantly reduced serum deprivation-induced apoptosis in cells transfected with AT1R(Del221/222). This study shows for the first time a direct link between kinase activation and inhibition of apoptosis dependent on amino acids 221 and 222 in the third cytoplasmic loop of the AT1.",
keywords = "Angiotensin II, Apoptosis, AT, Kinases",
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T1 - The third cytoplasmic loop of the angiotensin II type 1 receptor exerts differential effects on extracellular signal-regulated kinase (ERK1/ERK2) and apoptosis via Ras- and Rap1-dependent pathways

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AU - Ishida, Mari

AU - Hunyady, L.

AU - Berk, Bradford C.

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N2 - The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT1) is important for receptor coupling to G proteins and activation of downstream events. Therefore, we determined whether specific AT1 sequences were required for kinase activation and inhibition of apoptosis by transfecting wild-type (AT1Rwt) and mutated AT1 into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal-regulated kinase (ERK1/ERK2) activity in 293 cells transfected with AT1Rwt. However, in 293 cells that expressed a receptor in which amino acids 221 and 222 were deleted (AT1R[Del221/222]), Ang II-mediated ERK1/ERK2 activation was inhibited by >85%. In contrast, c-Jun NH2-terminal protein kinase (JNK) activation was similar in AT1Rwt- and AT1R(Del221/222)-transfected cells. Activation of ERK1/ERK2 by AT1Rwt was independent of Ca2+, whereas the low level of ERK1/ERK2 activation by AT1R(Del221/222) was completely Ca2+ dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(Del221/222) required Rap1. These results demonstrate the presence of 2 different pathways for ERK1/ERK2 activation by Ang II, which differ in their requirements for Ca2+ and small G proteins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation-induced apoptosis in cells transfected with AT1Rwt but not AT1R(Del221/222). AKT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpression of constitutively active AKT significantly reduced serum deprivation-induced apoptosis in cells transfected with AT1R(Del221/222). This study shows for the first time a direct link between kinase activation and inhibition of apoptosis dependent on amino acids 221 and 222 in the third cytoplasmic loop of the AT1.

AB - The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT1) is important for receptor coupling to G proteins and activation of downstream events. Therefore, we determined whether specific AT1 sequences were required for kinase activation and inhibition of apoptosis by transfecting wild-type (AT1Rwt) and mutated AT1 into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal-regulated kinase (ERK1/ERK2) activity in 293 cells transfected with AT1Rwt. However, in 293 cells that expressed a receptor in which amino acids 221 and 222 were deleted (AT1R[Del221/222]), Ang II-mediated ERK1/ERK2 activation was inhibited by >85%. In contrast, c-Jun NH2-terminal protein kinase (JNK) activation was similar in AT1Rwt- and AT1R(Del221/222)-transfected cells. Activation of ERK1/ERK2 by AT1Rwt was independent of Ca2+, whereas the low level of ERK1/ERK2 activation by AT1R(Del221/222) was completely Ca2+ dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(Del221/222) required Rap1. These results demonstrate the presence of 2 different pathways for ERK1/ERK2 activation by Ang II, which differ in their requirements for Ca2+ and small G proteins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation-induced apoptosis in cells transfected with AT1Rwt but not AT1R(Del221/222). AKT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpression of constitutively active AKT significantly reduced serum deprivation-induced apoptosis in cells transfected with AT1R(Del221/222). This study shows for the first time a direct link between kinase activation and inhibition of apoptosis dependent on amino acids 221 and 222 in the third cytoplasmic loop of the AT1.

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